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(1)City University of New York (CUNY). CUNY Academic Works Dissertations, Theses, and Capstone Projects. CUNY Graduate Center. 2-2019. Bisthioether Stapled Peptides Targeting Polycomb Repressive Complex 2 Gene Repression Gan Zhang The Graduate Center, City University of New York. How does access to this work benefit you? Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/2980 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: AcademicWorks@cuny.edu.

(2) Bisthioether Stapled Peptides Targeting Polycomb Repressive Complex 2 Gene Repression. By. Gan Zhang. A dissertation submitted to the Graduate Faculty in Chemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2019 i.

(3)  2019 Gan Zhang All Rights Reserved ii.

(4) Bisthioether Stapled Peptides Targeting Polycomb Repressive Complex 2 Gene Repression by Gan Zhang This manuscript has been read and accepted for the Graduate Faculty in Chemistry in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy.. Date. Guillermo Gerona-Navarro Chair of Examining Committee. Date. Brian R. Gibney Executive Officer Supervisory Committee: Maria Contel Ryan P. Murelli David R. Mootoo Lissette Delgado-Gruzata. THE CITY UNIVERSITY OF NEW YORK iii.

(5) Bisthioether Stapled Peptides Targeting Polycomb Repressive Complex 2 Gene Repression By Gan Zhang Adviser: Professor Guillermo Gerona-Navarro. Abstract Interactions between proteins play a key role in nearly all cellular process, and therefore, disruption of such interactions may lead to many different types of cellular dysfunctions. Hence, pathologic protein-protein interactions (PPIs) constitute highly attractive drug targets and hold great potential for developing novel therapeutic agents for the treatment of incurable human diseases. Unfortunately, the identification of PPI inhibitors is an extremely challenging task, since traditionally used small molecule ligands are mostly unable to cover and anchor on the extensive flat surfaces that define those binary protein complexes. In contrast, large biomolecules such as proteins or peptides are ideal fits for these so-called “undruggable” sites. However, their poor pharmacokinetic properties have limited their application as therapeutics. In this context, peptidomimetic molecules have emerged as an alternative and viable solution to this problem, since they conserve the architectural and structural features of peptides and also exhibit substantially improved pharmacokinetic profiles. Given the great promise of this class of compounds as therapeutics, new protocols granting easy access to them continue to be of great interest. This thesis describes the development of an efficient solid phase methodology for the chemoselective synthesis of bisthioether stapled peptides of multiple architectures and its application to discovering three families of potent allosteric inhibitors of the polycomb repressive complex 2 (PRC2) of proteins.. iv.

(6) PRC2 is a multimeric complex consisting of four core proteins: EZH2, EED, SUZ12 and RBAP46/RBAP48, which is involved in the initiation of gene repression through its methyltransferase activity, specific for lysine 27 on Histone H3 (H3K27). The enzymatic activity of PRC2 is conferred by the catalytic SET domain of EZH2, but also to the other core components of the complex. Hence, a catalytically active PRC2 complex must contain EZH2 and at least EED and SUZ12, which underscores the role of the latter proteins as scaffolds for the proper assembly of PRC2 into its bioactive conformation. The biological relevance of PRC2 proteins is highlighted by their known role in the development and progression of different types of cancers, and thus targeting them has emerged as a high-priority strategy in the field of cancer epigenetics. The thesis first describes the development of an innovative solid phase approach for the preparation of bisthioether stapled peptides of multiple architectures, including single-, doubleturn and double stapled peptides. This methodology allows for ligation with all-hydrocarbon linkers of various lengths, avoiding the use of unnatural amino acids and expensive catalysts, and affords cyclopeptides with improved bioactive conformation and remarkable resistance to proteolytic degradation. Next, we describe the rational design, synthesis and biological evaluation of three new families of allosteric inhibitors of PRC2 function, targeting for the first time three protein interfaces in PRC2 that are crucial for its proper assembly and function: the intramolecular SANT1L/SBD interaction of EZH2, the SUZ12-VEFS/EZH2-SANT2 binary complex and the interaction between SUZ12-NBE domains. Remarkably, these inhibitors have demonstrated cell permeability, potent activity in vitro and in physiological conditions, as well as strong antiproliferative effects on Caki-1 renal cancer cells, which highlights their potential as novel therapeutics for the treatment of PRC2-dependent human cancers.. v.

(7) DEDICATION. To my amazing parents, Lamei Xu and Jiangwei Zhang, for having always believed in me.. &. To my wonderful spouse, Dr. Tian Zhou for all the support and encouragement.. vi.

(8) Acknowledgements. I would like to express my sincere appreciation to everyone who has contributed their time and knowledge to make this thesis possible. I would first thank my advisor Professor Guillermo Gerona-Navarro, who accepted me during my hardest time, who has always trusted and given me these amazing projects and guided me to achieve the final success. I would like to thank all the members of my thesis committee, Professor Guillermo GeronaNavarro, Professor Maria Contel, Professor Ryan P. Murelli, Professor Lissette Delgado-Cruzata, Professor David R, Mootoo for their support and advice. I would like to thank Dr. Flavia Barragan for being a wonderful coworker and for all the training with lab instruments. I would like to thank undergraduate students who have contributed their effort for the projects in this thesis, Ms. Khadija Wilson, Mr. Adam Herskovits, Mr. Nissim Levy, Mr. Mikhail Menasherov, Mr. Haleem Alkasimi, Mr. Leutrim Kelmendi, Ms. Maisha M. Chowdhury, Ms. Milana Sapozhnikov, Ms. Garbielle A. DeSena. I would like to thank Prof. Yoel Rodriguez for performing molecular docking and modeling protein-peptide interactions. I would like to thank Professor Maria Contel, Dr. Benelita T. Elie, Mr. Mike Cornejo for performing cell culture and teaching me in vitro cell culture screening experiments. I would like to thank Professor Mariana Torrente, Ms. Karen Chen, Mr. Seth Bennett, Ms. Royena Tanaz for their training and instrument support of western-blot.. vii.

(9) I would like to thank Professor Aneta J. Mieszawska, Mr. Marek Wlodarczyk, Dr. Sylwia Dragulska, Ms. Mina Poursharifi for their training and instrument support of plate-reader and nanodrop. I would like to thank Professor Maggie Ciszkowska for her advice and support throughout these years. I would like to thank Chemistry Department at Brooklyn College for all the help, and Ph.D. Program in Chemistry at Graduate Center, City University of New York for giving me the opportunity for this amazing journey. I would like to thank the financial support from National Institute of Health (NIH) 5SC2GM111231-02. I would like to thank all my friends Dr. Luwen (Andy) Chao, Ms. Suiying Huang, Dr. Malgorzata Frik, Dr. Michael D’resmo and Dr. Danille Hirsh for their support throughout the years. And finally, I would like to thank myself for staying determined and strong to accomplish this wonderful task. I will always believe in myself.. viii.

(10) 1. CHAPTER I. PEPTIDOMIMETICS AS CHEMICAL PROBES TO TARGET. INTRACELLULAR PROTEIN-PROTEIN INTERACTIONS ............................................... 1 1.1. BACKGROUND AND SIGNIFICANCE ................................................................................... 1. 1.2. STAPLED PEPTIDES .......................................................................................................... 2. 1.2.1. Synthetic Methods Yielding Stapled Peptides ............................................................ 4. 1.2.2. Cell Permeability of Stapled Peptides....................................................................... 12. 1.2.3. Staple Peptide’s Stability to Proteolysis ................................................................... 14. 1.2.4. Stapled Peptides: Improved Target Binding Affinity and Diversity of Binding. Strategies ............................................................................................................................... 15 1.3. LONG RANGE MACROCYCLIZATION METHODOLOGIES .................................................. 19. 1.3.1. Cell Permeability of Macrocyclic Peptides............................................................... 20. 1.3.2. Macrocyclic Peptides as inhibitors of PPIs ............................................................... 21. 1.4. PEPTIDOMIMETIC MOLECULES FOR THERAPEUTIC DEVELOPMENT: SIGNIFICANCE ........ 22. 1.5. ACRONYMS AND ABBREVIATIONS ................................................................................. 24. 2. CHAPTER II. A SOLID PHASE APPROACH TO ACCESSING BISTHIOETHER. STAPLED PEPTIDES................................................................................................................ 26 2.1. INTRODUCTION .............................................................................................................. 26. 2.2. OPTIMIZATION OF THE STAPLING REACTION.................................................................. 26. 2.3. SOLID PHASE SYNTHESIS OF SINGLE-TURN STAPLED PEPTIDES (I, I+4). ....................... 30. 2.4. SOLID PHASE SYNTHESIS (SPPS) OF DOUBLE-TURN STAPLED PEPTIDES (I, I+7) .......... 35. 2.5. SOLID PHASE SYNTHESIS OF SINGLE-TURN STAPLED PEPTIDES WITH ADDITIONAL. CYSTEINES IN THE SEQUENCE (I, I+4, +CYS). ............................................................................ 36. 3. 2.6. SOLID PHASE SYNTHESIS OF DOUBLE STAPLED PEPTIDES [2 X (I, I+4)]......................... 38. 2.7. CONCLUSION.................................................................................................................. 40. 2.8. EXPERIMENTAL SECTION ............................................................................................... 40. 2.8.1. Acronyms and Abbreviations ................................................................................... 40. 2.8.2. General experimental information ............................................................................ 41. 2.9. APPENDIX: SUPPLEMENTARY FIGURES .......................................................................... 49. 2.10. APPENDIX: PEPTIDE’S CHARACTERIZATION ................................................................... 57 CHAPTER III. STAPLED PEPTIDE INHIBITORS POLYCOMB REPRESSIVE 2. GENE REPRESSION ................................................................................................................. 67 ix.

(11) 3.1. BACKGROUND................................................................................................................ 67. 3.2. BISTHIOETHER STAPLED PEPTIDES AS ALLOSTERIC INHIBITORS OF PRC2 TARGETING. THE SANT1L-SBD INTERACTION IN EZH2. ............................................................................. 70. 3.3. BISTHIOETHER STAPLED PEPTIDES AS ALLOSTERIC INHIBITORS OF THE SUZ12-. VEFS/EZH2-SANT2 INTERACTION.......................................................................................... 82 3.4. BISTHIOETHER STAPLED PEPTIDES AS ALLOSTERIC INHIBITORS OF THE SUZ12-. NBE/NURF55 DOMAIN INTERACTION........................................................................................ 90 3.5 3.5.1. General experimental information .......................................................................... 101. 3.5.2. Biochemical and Cellular Studies ........................................................................... 103. 3.6 4. APPENDIX ................................................................................................................. 109 CHAPTER IV. SUMMARY ........................................................................................ 129. 4.1 5. EXPERIMENTAL SECTION .................................................................................... 101. SUMMARY .................................................................................................................... 129 BIBLIOGRAPHY ......................................................................................................... 131. x.

(12) Figure 1.2.1 The stapled peptide approach (A) Single turn stapled peptide (i, i+4) (B) Double turn stapled peptide (i, i+7). ................................................................................................................... 3 Figure 1.3.1 Structural representation of long range macrocyclization. ...................................... 19 Figure 3.2.1 Bis-thioether Stapled Peptide Design Left: The crystal structure of an active Ezh2Eed-Suz12(VEFS) PRC2 complex ortholog from yeast (PDB ID 5KJH)99 revealed key interactions for PRC2 catalytic structure and function. Top Right: Zoomed alpha-helical intramolecular interaction in between the SBD (colored gray) and SANT1L (colored orange) EZH2 domains. Bottom Right: Structure of the designed stapled peptide inhibitors targeting the SBD-SANT1L interaction in EZH2, using the same color coding. All cyclopeptides are derived from the SBD wild type sequence 1, residues 198-216 in EZH2. Solvent-exposed amino acids were mutated to Cys for peptide stapling (colored pink for single- and yellow for double-turn stapling). ............ 71 Figure 3.2.2 Biochemical Assays (a) Structure of the synthesized stapled peptides, and their correspondent helicity and IC50 values for inhibition of H3K27 trimethylation in vitro, as determined by a histone methyltransferase colorimetric assay optimized using endogenous PRC2 extracted from a human clear cell renal carcinoma cell line (Caki-1). (b) CD spectra of stapled peptides and their linear counterparts measured in water at 20 oC. (c) Plot of H3K27 trimethylation inhibition using the enzymatic in vitro assay described in a, with varying concentrations of 2, positive control GSK126, and using an equimolar combination of both compounds to test their synergistic effects. The IC50 values obtained when using recombinant PRC2 and when using a high concentration of SAM (110 X) are also shown. A summary of the IC 50 values for inhibition of H3K27me3 determined from the experiments shown in plot c. is also presented. Calculation of the combination index using the equation described by Chou et al. (CI= 0.53) indicated a marked synergistic effect between 2 and GSK126. ................................................................................... 75 Figure 3.2.3 (a) Structure of the biotinylated derivative of cyclopeptide 2, synthesized on solid phase (b,c) In vitro pull down assay using biotinylated cyclopeptide 2, visualized by SDS-PAGE and followed by western blot analysis using antibodies against human EZH2 (b) and human EED and SUZ12 (c). .............................................................................................................................. 77 Figure 3.2.4 The Chymotrypsin-based proteolytic degradation assays as well as the plasma stability assay show remarkable stability of our i, i+4 and i, i+7 bisthioether stapled peptides. The rate of peptide proteolysis was monitored by HPLC and HPLC-MS analysis. ............................ 78 Figure 3.2.5 Confocal microscopy images of Caki-1 cells, scale bar 10 m (a) Cells were treated with 5M of FITC-labeled 13 at 37oC for 5h. 60X magnification (b) Vehicle, cells were treated with 1% dmso in PBS at 37oC for 5h. 60X magnification............................................................ 79 Figure 3.2.6 Cellular Assays with lead bisthioether stapled peptide 2 (a) Western blot analysis shows dose-dependent response of H3K27me3, H3K27me2 and H3K27me1 within caki-1 cells, after treatment with stapled peptide 2 once daily for 72h. Single concentration treatment with GSK126 (positive control) and linear wild type sequence (1, negative control) in the same experimental conditions are also shown. Quantitation of H3K27me3 using and absorbance-based colorimetric assay yielded an IC50 value of 0.4  0.2 M. Protein loading was accurately corrected by measuring total H3 using an absorbance-based colorimetric assay. (b) Selectivity of H3K27 trimethylation inhibition over a broad panel of histone post-translational modifications. Cells were treated with stapled peptide 2 (5 M) or vehicle, once daily for 72h. (c) Treatment of Caki-1 cells with cyclopeptide 2, control compound GSK126, and an equimolar combination of both molecules significantly inhibits cell proliferation. Proliferation was measured after 72h of daily treatment with the correspondent compound. The data is presented as a mean of two independent experiments each with triplicate measurements. (d) Plot of IC50 values obtained when testing our xi.

(13) lead compound 2, GSK126 and a combination of both in: (d.1) an enzymatic in vitro assay monitoring H3K27me3 inhibition, using endogenous PRC2 extracted from a human clear cell renal carcinoma cell line (Caki-1), (d.2) a proliferation colorimetric assay with Caki-1 cells, (d.3) in non-cancerous human fibroblast IMR90 cells. Calculation of the combination index using the equation described by Chou et al. indicated a marked synergistic antiproliferative effect (CI= 0.61) when both compounds are used together. ..................................................................................... 80 Figure 3.3.1 Left: The cryo-electron microscopy structure of a complete human PRC2 complex (PDB ID 6C23)98. Top Right: Zoomed -helical intramolecular interaction between the targeted VEFS-SUZ12 (colored purple, yellow and gray) and EZH2 domains (colored Cyan). Bottom Right: Structure of the designed stapled peptide inhibitors, with the Cys residues incorporated for stapling labelled in green. ........................................................................................................................... 83 Figure 3.3.2 (a) Structure of the designed stapled peptides and summary of results obtained in the biochemical assays (b) Both Chymotrypsin-based proteolytic degradation and the plasma assay show enhanced stability of i, i+4 bisthioether stapled peptides. (c) CD spectra of stapled peptides and their linear counterparts measured in water at 20 oC. (d) Inhibition of PRC2 catalytic function (H3K27Me3) as determined in an enzymatic assay using endogenous PRC2 extracted from a human clear cell renal carcinoma cell line (Caki-1), at peptide concentration of 10 M (e) Plot of H3K27me3 inhibition obtained in an enzymatic assay using endogenous PRC2 extracted from Caki-1 cells and recombinant PRC2 complex, 100SAM, and equimolar combination with GSK126 (for compound 11). ........................................................................................................ 86 Figure 3.3.3 (a) Confocal microscopy images of Caki-1 cells, scale bar 10 m. Cells were treated with FITC-labeled 11 (5 M) and control peptide (-Ala--Ala-FITC, 5 M) at 37oC for 5h. (b) Western blotting after treating caki-1 cells with both series of peptides at 10 M shows marked inhibition of H3K27me3 by compound 11, in contrast to the control peptides (c) Western blot analysis after treatment of caki-1 cells with varying concentration of stapled peptide 11, once daily for 72h, indicates a clear dose-dependent response of H3K27me3. Single concentration treatment with GSK126 (positive control) and linear wild type sequence 16 (negative control) are also shown. (d) Selectivity of H3K27 trimethylation inhibition over a broad panel of histone post-translational modifications. Cells were treated with stapled peptide 11 (5 M) or vehicle, once daily for 72h. ....................................................................................................................................................... 89 Figure 3.4.1 Top: Crystal structure of Nurf55 in complex with SUZ12-NBE (PDB 2YB8)119. Center: Zoomed -helical intramolecular interaction between Nurff55 and the Nurf55 binding epitope (NBE) domain of SUZ12 (SUZ12-NBE). Bottom: Structure of the designed stapled peptide inhibitors, mimetics of the helix α1 of Nurf55, residues 20-35, with the Cys residues incorporated for stapling labelled in purple. ................................................................................. 91 Figure 3.4.2 (a) Structure, helicity and % of inhibition of H3K27me3 at 10 M for all the synthesized stapled peptides (b) Bar graph showing the helicities and percentages of H3K27me3 inhibition at 10 M peptide concentration, for all the synthesized stapled peptides and the linear analogue 13 (c) CD spectra of each of the peptides that showed the higher helical character from each family, measured in water at 20oC........................................................................................ 94 Figure 3.4.3 (a) Inhibition of PRC2 catalytic function (H3K27Me3) by the most potent stapled peptides of each series, as determined in an enzymatic assay using endogenous PRC2 extracted from a human clear cell renal carcinoma cell line (Caki-1) (b) Plot of H3K27 trimethylation inhibition using the enzymatic in vitro assay described in a (c) Plot of H3K27 trimethylation inhibition using the enzymatic in vitro assay described in a, with varying concentrations of 24 (using both endogenous PRC2 extracted from a human clear cell renal carcinoma cell line and xii.

(14) recombinant PRC2), positive control GSK126, and using an equimolar combination of both compounds to test their synergistic effects. (d) Plot of H3K27 trimethylation inhibition using the enzymatic in vitro assay described in a, with varying concentrations of SAM. (e) A summary of the IC50 values for inhibition of H3K27me3 determined from these experiments is shown in plot b,c and d. ...................................................................................................................................... 95 Figure 3.4.4 (a) Structure of the biotinylated derivative of cyclopeptide 24, synthesized on solid phase (b,c) In vitro pull down assay using biotinylated cyclopeptide 2, visualized by SDS-PAGE and followed by western blot analysis using antibodies against human EZH2 (b) and human EED and SUZ12 (c). .............................................................................................................................. 96 Figure 3.4.5 Both Chymotrypsin-based proteolytic degradation and the plasma assay show enhanced stability of our i, i+4 bisthioether stapled peptide 24, as compared to its linear parent peptide 13. ..................................................................................................................................... 97 Figure 3.4.6 Confocal microscopy images of Caki-1 cells, scale bar 10 m. Cells were treated with FITC-labeled 24 (24-FITC, 5 M) and control peptide (-Ala--Ala-FITC, 5 M) at 37oC for 5h. ............................................................................................................................................ 98 Figure 3.4.7 (a) Western blot analysis shows dose-dependent response of H3K27me3 within metastatic human clear-cells renal carcinoma cells (Caki-1), after treatment with stapled peptide 24 once daily for 72h. Single concentration treatment with GSK126 (positive control) and linear wild type sequence 13 (negative control) in the same experimental conditions are also shown. (b) Quantitation of H3K27me3 using and absorbance-based colorimetric assay yielded an IC50 value of 1.36  1.14 M. Protein loading was accurately corrected by measuring total H3 using an absorbance-based colorimetric assay. (c) Selectivity of H3K27 trimethylation inhibition over a broad panel of histone post-translational modifications. Cells were treated with stapled peptide 24 (5 M) or vehicle, once daily for 72h. (d) Treatment of Caki-1 cells with cyclopeptide 24 significantly inhibits cell proliferation. Proliferation was measured after 72h of daily treatment with the correspondent compound. The data is presented as a mean of two independent experiments each with triplicate measurements. ........................................................................ 100. xiii.

(15) Figure S 2.1 HPLC analysis of the crude reaction mixtures obtained after cleavage of the stapling reactions for the (i, i+4) stapled peptides 2-5, and their correspondent linear precursor 1. Experimental conditions used for each HPLC analysis are given in the peptide characterization section. .......................................................................................................................................... 49 Figure S 2.2 HRMS analysis of crude reaction mixtures obtained after cleavage of the stapling reactions for the (i, i+4) stapled peptides 2-5 and their correspondent linear precursor 1. Experimental conditions used for each HRMS analysis are given in the general experimental procedure section. Observed 121.05 Da and 922.01 peaks correspond to internal references used in the analysis. ............................................................................................................................... 50 Figure S2.3 HPLC analysis of the crude reaction mixtures obtained after cleavage of the stapling reactions for the (i, i+7) stapled peptides 7-9 and their correspondent linear precursor 6. Experimental conditions used for each HPLC analysis are given in the peptide characterization section. .......................................................................................................................................... 51 Figure S 2.4 HRMS analysis of crude reaction mixtures obtained after cleavage of the stapling reactions for the (i, i+7) stapled peptides 7-9 and their correspondent linear precursor 6. Experimental conditions used for each HRMS analysis are given in the general experimental procedure section. Peaks observed at 121.05 Da and 922.01 Da in the HRMS spectra correspond to internal references used in the analysis..................................................................................... 52 Figure S 2.5 HPLC analysis of the crude reaction mixtures obtained after cleavage of the stapling reactions for the (i, i+4) stapled peptides containing an additional Cys residue 10-12 and their correspondent linear precursor S2a. Experimental conditions used for each HPLC analysis are given in the peptide characterization section. ............................................................................... 53 Figure S 2.6 HRMS analysis of crude reaction mixtures obtained after cleavage of the stapling reactions for the (i, i+4) stapled peptides containing an additional Cys residue 10-12 and their correspondent linear precursor S2a. Experimental conditions used for each HRMS analysis are given in the general experimental procedure section. Observed 121.05 Da and 922.01 peaks correspond to internal references used in the analysis. ................................................................. 54 Figure S 2.7 HPLC analysis of the crude reaction mixtures obtained after cleavage of the second stapling reaction for the double stapled peptide [2 x (i, i+4)] 14 and its correspondent linear precursor 13. Experimental conditions used for each HPLC run are given in the peptide characterization section. ................................................................................................................ 55 Figure S 2.8 HRMS analysis of crude reaction mixtures obtained after cleavage of the first and second stapling reactions for the double stapled [2 x (i, i+4)] 14 and its correspondent linear precursor 13. Experimental conditions used for each HRMS analysis are given in the general experimental procedure section. Observed 121.05 Da and 922.01 peaks correspond to internal references used in the analysis. ..................................................................................................... 56. xiv.

(16) Table 1.1 Summary of Stapling Methodologies........................................................................... 10 Table 1.2 Applications of the Stapled Peptide Technology in a Variety of Human Diseases ..... 18 Table 1.3 Representative examples of macrocyclic peptides disrupting biologically relevant PPIs. ....................................................................................................................................................... 22 Table 2.1 Preliminary optimization results of solid phase cyclization using a 10 mer peptide (Scheme 2.1): Resin-ISLISCSLSC-Alloc (Optimal results obtained with conditions described in entry 7J) ........................................................................................................................................ 29 Table 2.2 Synthesis of single- (i,i+4), double-turn (i,i+7) and double-stapled peptides ............. 33. xv.

(17) Scheme 1.2.1 Schematic representation for the synthesis of stapled peptides using the olefin metathesis reaction. Incorporation of -methyl, -alkenyl amino acids followed by stapling through ring closing metathesis affords all-hydrocarbon stapled peptides of various architectures. ......................................................................................................................................................... 4 Scheme 1.2.2 Solution phase approaches for the synthesis of bisthioether stapled peptides using highly reactive dihaloelectrophiles. ................................................................................................ 5 Scheme 1.2.3 Solution synthesis of bisthioether stapled peptides by means of a two-component thiol-ene reaction between a fully deprotected peptides and various dienes. ................................. 6 Scheme 1.2.4 Solution synthesis of bis-selenoether stapled peptides by means of a two-component reaction between fully deprotected selenocysteine-substituted peptides and various electrophiles. ......................................................................................................................................................... 7 Scheme 1.2.5 (a) Peptide stapling through lactamisation, a polar amide bond can be chemoselectively formed by orthogonally protecting both the amino and the carboxylate functional groups used for the macrocyclization (b) Nitrogen arylation of unprotected peptides allows crosslinking through lysine-based nucleophiles, using highly reactive aryl electrophiles. .................... 8 Scheme 1.2.6 (a) Cu(I)-catalyzed cycloaddition between bis-alkynes and a bis-azide functionalized peptide allows for peptide cross-linking through a bis-triazole linker (b) SuzukiMiyaura coupling affords stapled peptides with a biphenyl ring spacer. ....................................... 9 Scheme 2.2.1 Synthetic scheme initially designed to study the stapling reaction on a 10mer linear Peptide........................................................................................................................................... 28 Scheme 2.3.1 Solid phase synthesis of single-turn (i, i+4) stapled peptides bearing all-hydrocarbon linkers of different lengths. ........................................................................................................... 31 Scheme 2.3.2 Maleimide test developed to assess the efficiency of the Mmt-deprotection reaction. Since bulky protecting groups in the vicinity of Cys-Mmt could cause incomplete deprotection despite the sensitivity of Mmt to acid media, we developed conditions for quantitatively assessing the efficiency of Mmt deprotection. Free thiols generated after deprotection, are covalently blocked through a well-known thiol-maleimide addition step. Cleavage of an aliquot of the reacted peptide, and LC/MS analysis allow to unequivocally assign fully deprotected products, partially or non-deprotected sequences, as well as mixtures containing all of them. .................................................................................................................................... 32 Scheme 2.3.3 Mechanism for the formation of bis-dehydroalanine substituted peptides as a side product in the direct alkylation of cysteine residues. Cysteine conversion to dehydroalanines (Dha) was recently reported by Chalker et al. to take place through a bis-alkylation-elimination mechanism, which is particularly effective when using 1,4-diodo or 1,4-dibromobutane as electrophiles. ................................................................................................................................. 34 Scheme 2.4.1 Solid phase synthesis of single-turn (i, i+7) stapled peptides bearing all-hydrocarbon linkers of different lengths. Full characterization and experimental details are given in the Appendixes. .................................................................................................................................. 36 Scheme 2.5.1 Synthetic scheme for the preparation of single-turn stapled peptides containing an additional cysteine residue in the sequence (i, i+4, +Cys). .......................................................... 38 Scheme 2.6.1 Synthetic scheme for the chemoselective preparation of double-turn stapled [2 x (i, i+4)]. ............................................................................................................................................. 39 Scheme 3.2.1Synthetic scheme for the chemoselective preparation of single turn stapled peptides (i, i+4) inhibitors of the intramolecular SANT1L-SBD interaction in EZH2. ............................. 72 Scheme 3.2.2 Synthetic scheme for the chemoselective preparation of double turn stapled peptides (i, i+7) inhibitors of the intramolecular SANT1L-SBD interaction in EZH2. ............................. 73 xvi.

(18) Scheme 3.3.1 Synthetic scheme for the preparation of single turn stapled peptides (i, i+4) mimetics of the SUZ12-VEFS domain, residues 590-603, as inhibitors of PRC2 methyltransferase activity. ....................................................................................................................................................... 84 Scheme 3.3.2 Synthetic scheme for the preparation of single turn stapled peptides (i, i+4) mimetics of the SUZ12-VEFS domain, residues 652-669, as inhibitors of PRC2 methyltransferase activity. ....................................................................................................................................................... 85 Scheme 3.4.1 Synthetic scheme for the preparation of single turn stapled peptides 24-28 (i, i+4), as mimetics of the helix α1 of Nurf55, residues 20-35, targeting the SUZ12-NBE domain. ....... 92. xvii.

(19) Chapter I. Peptidomimetics as Chemical Probes to Target Intracellular Protein-Protein Interactions. 1.1. Background and Significance Protein-protein interactions (PPIs) are crucial for a plethora of cellular processes and thus. for the proper functioning of living cells and organisms. Their dysregulation can lead to many types of cellular dysfunctions which can further develop into diseases, hence, targeting specific PPIs is currently one of the most attractive approaches for designing potential therapeutics 1-4. The “undruggable” nature of these attractive macromolecular interactions, however, have made them particularly challenging targets. PPIs involve large, flat and featureless surfaces, ranging from 1500 to 3000 A2 and lacking well-defined architectural features such as hydrophobic pockets or clefts5-7. Thus, these recognition events result from a collection of scattered smaller interactions or hot-spots, which are structurally unsuitable for small molecules ligands, traditionally used by medicinal chemists8-11. Indeed, effectively disrupting PPIs requires the use of larger molecules, capable of covering extensive molecular areas and establishing contacts with multiple hot spots simultaneously. Peptides are great candidates for such purposes since they are large enough to cover lengthy contact surfaces and also have the potential to recapitulate the shape and conformation of natural binding sites12-14. In addition, they are easily accessible, offer high structural diversity and low toxicity. Unfortunately, high susceptibility to protease degradation and often poor cell permeability and binding affinities, have limited their therapeutic applications, especially for targeting intracellular PPIs15-16. 1.

(20) Extensive efforts have been made to overcome the poor pharmacokinetic properties of peptides and to transform them into more ideal drug candidates11-14, 17. As a result of this work, several approaches yielding peptidomimetic molecules have been discovered. These synthetically modified peptides, with improved pharmacokinetic profiles, are designed to mimic their bioactive parent sequences and have encountered applications in a wealth of biological targets, showing great promise as potential modulators of undruggable intracellular PPIs 11, 13-15. Several reviews in the literature have discussed the progress in the field, primarily focusing on either specific families of compounds or in their biological applications11-18. Since the number of relevant reports covering this attractive research area continues to grow, we present here a wider comprehensive updated overview focused on both the synthesis and the biological evaluation of two major groups of biologically active peptidomimetic derivatives: “stapled peptides”, designed to mimic alpha helical motifs of protein domains; and cyclopeptides resulting from long range macrocyclizations, aimed at mimicking beta sheets and other turn motifs. Some relevant biophysical properties of such compounds, including their cellular uptake and proteolytic stability are discussed as well.. 1.2. Stapled Peptides Peptide stapling has emerged as one of the most successful strategies to produce potent. peptidomimetic inhibitors of intracellular PPIs18-20. This methodology aims primarily at mimicking native-helical domains, a recurrent protein motif that serve as a fundamental recognition unit in over 40% of naturally occurring PPIs. The idea behind stapling a peptide lies in tethering the side chain of a residue i with that of another residue at position i+4 or i+7 in the sequence, since these residues protrude on the same face of the helix (Figure 1.2.1). By incorporating a covalent “staple” the peptide is induced into its bioactive -helical conformation, 2.

(21) that in addition, is stabilized by intramolecular hydrogen bonding between the amide bonds on opposite sides of the helix21. This conformational restriction effectively improves not only the helical character of a given peptide sequence, and thus, its binding affinity to the protein target, but also its resistance to proteolysis and cell permeability properties 22-23. Several factors impact the extent of such improvement, particularly the length of the peptide sequence, as well as the position and physicochemical properties of the staple. A series of chemical strategies to accomplish peptide stapling have been reported to date. Next, we summarize them and further discuss the pharmacokinetic properties of the corresponding likers used for ligation, as well as some examples demonstrating the wide range of biological applications for which this promising new class of compounds have been successfully applied.. (i, i + 4) Linker. (i, i + 7) Linker. Figure 1.2.1 The stapled peptide approach (A) Single turn stapled peptide (i, i+4) (B) Double turn stapled peptide (i, i+7).. 3.

(22) 1.2.1 Synthetic Methods Yielding Stapled Peptides The incorporation of all-hydrocarbon staples by means of the ring-closing metathesis (RCM) reaction is the most widely used method for stabilizing α-helical peptides. This approach was first developed by Blackwell and Grubbs, who established an efficient solid phase procedure for the macrocyclization of linear peptides containing O-allylserine residues, via the rutheniumcatalyzed RCM reaction24. An extension of this work by Verdine and colleagues further allowed for ligation of alkenyl side chains of ,-disubstituted unnatural amino acids, previously incorporated at positions (i, i+3), (i, i+4) and (i, i+7) in the corresponding linear sequences, under the same RCM stapling conditions25 (Scheme 1.2.1). This latter methodology constituted a major breakthrough in the field since it yielded macrocyclic -helical peptides with remarkable resistance to proteolytic degradation, significantly improved alpha-helical character and enhanced affinity to their protein targets. Verdine’s pioneer stapling technology has been applied to generate multiple all-hydrocarbon stapled peptide inhibitors of intracellular PPIs with a wide variety of biological applications22, 26-28. Moreover, it has been recently applied to generate the first stapled peptide drugs entering clinical trials29. i. i. i+7. Cl Cl. i+3. PCy3 PCy3. n. Cl Cl. n. Resin. PCy3 PCy3 i. i+4. Scheme 1.2.1 Schematic representation for the synthesis of stapled peptides using the olefin metathesis reaction. Incorporation of -methyl, -alkenyl amino acids followed by stapling through ring closing metathesis affords all-hydrocarbon stapled peptides of various architectures.. 4.

(23) Several other synthetic approaches allow for peptide stapling using alternative chemistries and linkers with diverse topologies, including ligation of cysteine-thiols, disulfide bridges, lactamization reactions, nitrogen arylation, formation of oxime or hydrazones linkers, Cu(I) catalyzed azide-alkyne cycloadditions and metal-catalyzed arylation reactions30. The ligation of cysteine residues exploits the nucleophilicity of the thiol functional group to facilitate substitution reactions over highly reactive electrophiles, affording bisthioether stapled peptides tethered mainly by different aromatic ring scaffolds31-32. Several bis-aromatic electrophiles have been successfully used for this purpose, including photosensitive 4,4’bis(iodoacetamide)azobenzene33-34’-dibromo-m-xylene35-36,. 4,4’-bis(chloroacetamide). biphenyl-acetyl-ene37 and 4,4’-bis(bromomethyl)biphenyl (Scheme 1.2.2)38. Other approaches have used 1,3-dichloro-acetone39, perfluoaryl40, tetrazine41 and bis-palladium aryl derivatives42.. Linker S. i. S. SH. i+4. NH4HCO3 (20mM, pH = 8) Y-Linker-Y (Y= Cl, Br, I) R.T.. S. i. NH4HCO3 (20mM, pH = 8). SH. Linker. S. i+7. Y-Linker-Y (Y= Cl, Br, I) R.T.. Linkers: R. R. O. N N. H N O. H N n. O n = 1 to 10. H N O. O. H N n. N N. O. N N. HN. O. HN. O S O. N. NH. O NH O. n = 2 to 3. N. O O. O. O. S. O O. O. O. O S O NH. HN O. S O O. O. Scheme 1.2.2 Solution phase approaches for the synthesis of bisthioether stapled peptides using highly reactive dihaloelectrophiles.. On the other hand, only a small number of protocols allow cysteine ligation with allhydrocarbon linkers, primarily due to the poor electrophilicity of aliphatic dihaloalkanes. Thus,. 5.

(24) for example, Jo et al. reported that the direct bis-alkylation of cysteine thiols with dibromo and diiodoalkanes in solution phase only affords the corresponding unreacted linear precursors mixed with disulfide-bridged peptides35. An extension of this solution protocol including TCEP as reducing agent, longer reaction times and higher temperatures has allowed the preparation of i+4 and i+7 bisthioether stapled peptides linked through eight and nine methylene groups, respectively43. A similar solution phase approach, using commercially available 3,3bis(bromomethyl)oxetane as electrophile, has been recently applied to the efficient stapling of cysteines residues on peptides and proteins under biocompatible aqueous conditions 44. An elegant alternative to the two-component bis-alkylation of cysteine thiols with poorly electrophilic aliphatic cross-linkers has been recently reported by Wang et al., who have applied a photoinduced thiol-ene radical reaction with ,-dienes to produce bis-thioether stapled macrocycles bearing hydrocarbon braces with five, seven and nine methylene groups (Scheme 1.2.3)45. Linker i. S. S. SH. i+4. Diene Radical Initiator. SH. NMP, hv, 365 nm, 15 min. Diene Radical Initiator. i. S. Linker. S. i+7. NMP, hv, 365 nm, 15 min. Linkers: CO2H n n= 0, 2,3,4. O. O O. O. O. O. O. O. Scheme 1.2.3 Solution synthesis of bisthioether stapled peptides by means of a two-component thiol-ene reaction between a fully deprotected peptides and various dienes.. Although this solution protocol avoids using unnatural amino acids, its scope is somewhat limited by its required use of a high boiling point organic solvent, UV-light activation and a radical. 6.

(25) initiator as reaction catalyst. Thiol/yne stapling can also be carried out through a similar onecomponent radical reaction with alkynes, affording vinyl sulfide linked cyclopeptides 46.. Recently, the more reactive selenocysteine (as compared to cysteine) unnatural amino acids have been successfully used for peptide stapling through a selenoether linkage. This solution phase protocol allows for ligation with a wide range of alkylating reagents including poorly electrophilic dihaloalkanes (Scheme 1.2.4)47. Likewise, stapling can also be achieved by means of disulfide bridges48-49. Indeed, this was one of the first reported ligation techniques. However, due to the instability of the disulfide linkage in reducing environments this method has encountered only limited application. A recent example uses a reversible reaction for stapling BID and RNase S modified peptides through either oxidized disulfides or a dibromomaleimide crosslinker 50. The resulting macrocycles show resistance to proteolysis, enhanced -helical conformation and improved biological activity as compared to their respective linear counterparts. SeH SeH. Linker i. Se. Se. Linkers: n. i+4. Dibromolinker, DTT DMF:buffer pH 6-8.5 (1:1). Se. Linker. Se. i + 11. Linkers:. n= 1, 2, 5, 8, 10 i. O. i. Se Linker Se. i+7. n= 2, 3 n. Scheme 1.2.4 Solution synthesis of bis-selenoether stapled peptides by means of a two-component reaction between fully deprotected selenocysteine-substituted peptides and various electrophiles.. 7.

(26) Lactamisations have also been extensively applied for peptide stapling at positions (i, i+4) through amide-bond forming intramolecular cyclization of, for example, Lys/Asp, Orn/Lys and Asp/Glu residues (Scheme 1.2.5a)51-53. An advantage of this approach is that it uses natural amino acids, however, it also requires orthogonal protection of the residues participating in the ligation step. Nonetheless, several groups have applied this technique to generate cyclopeptides with superior -helicity and biological activity, mostly targeting extracellular or membrane-bound PPIs. More recently, lysine-based substituted peptides have also been used as scaffolds to generate aryltethered stapled peptides, through an N-arylation intramolecular macrocyclization and using several aromatic bis-electrophiles (Scheme 1.2.5b)54.. O NH2 n. HN COOH n. BOP DIPEA. n. n. Resin. Ar. Ar HN. NH. NH2 Ar. NH2. X X Aromatic Electrophile X = F, Cl Base DMF. Ar. HN. NH. X X Aromatic Electrophile X = F, Cl Base DMF. Scheme 1.2.5 (a) Peptide stapling through lactamisation, a polar amide bond can be chemoselectively formed by orthogonally protecting both the amino and the carboxylate functional groups used for the macrocyclization (b) Nitrogen arylation of unprotected peptides allows crosslinking through lysine-based nucleophiles, using highly reactive aryl electrophiles.. The well-known click cycloaddition between an azide and an alkyne is another example of a widely-used organic reaction applied to this field. This solution phase approach requires the incorporation of two azide functionalized amino acids at positions (i, i+6), (i, i+7) or (i, i+8) in the linear sequence. The ligation is next achieved by means of a double-click stapling step with 8.

(27) dialkynyl linkers under Cu(I) catalysis, affording bis-triazole tethered peptides (Scheme 1.2.6)28, 55-58. . Similarly, peptide stapling has also been accomplished by popular metal catalyzed C-C bond. formation reactions. Thus, using the Suzuki-Miyaura methodology Meyer and colleagues have prepared bi-aryl linked cyclopeptides, from linear sequences containing borylated phenyalanine residues59. Likewise, Mendive-Tapia et al. have reported an intramolecular cross coupling ligation between iodinated phenylalanine or tyrosine precursors and tryptophan residues, through a C-H activation process catalyzed by Pd(II). This approach yields aryl-tryptophan linked macrocycles of various sizes and is carried out in both solution and on solid phase 60.. N. R. N. N. N3 n. N n. R. N3. N N. n. n. Cu (I), Ligand (THPTA) ACN/H2O, (1:1) R.T. 16 hrs Y Y. I,Br I,Br. Z. Z. Pd(OAc)2, dppf Dioxane, CsF, H2O, 90 oC Suzuki-Miyaura. Y= H, Cl or OMe Z= H, OH or OMe. Scheme 1.2.6 (a) Cu(I)-catalyzed cycloaddition between bis-alkynes and a bis-azide functionalized peptide allows for peptide cross-linking through a bis-triazole linker (b) SuzukiMiyaura coupling affords stapled peptides with a biphenyl ring spacer.. 9.

(28) Table 1.1 Summary of Stapling Methodologies. 10.

(29) Overall, the number of approaches applied to stabilize peptide -helical structures has expanded significantly in the last few years and continues to grow fast. The versatility of these methods has undoubtedly facilitated the identification of multiple bioactive cyclopeptides, particularly useful to target intracellular intractable PPIs. Each of them has their own strengths but they also have some associated weaknesses. For example, most protocols require the use of expensive unnatural amino acids which increases significantly the overall cost of the process. An exception to this problem is to exploit the reactivity of native cysteine residues. Other significant drawbacks include the need for metal catalysis, high boiling point organic solvents (for solution phase methods) and photo-induced activation as in the case of the thiol-ene/yne ligations. Another major difference comes from the topology of the staple resulting from the ligation step, which may have a significant impact on the induction of the -helical conformation of the macrocycle. This feature has been recently explored by De d. Araujo et al. who tested the degree of helical induction in a model pentapeptide ligated with six different linkers 61. These studies showed that lactamisation led to the greatest increase in -helicity closely followed by the all-hydrocarbon staple and triazole spacers, whereas mono- and bis-thioether linkages showed lower ability to stabilize the peptide -helical conformation. More comprehensive studies in larger macrocycles and using other stapling chemistries will provide additional valuable insights into this critical factor for stapled peptide’s design. In summary, the field of stapled peptide synthesis has attracted significant interest since the discovery of the RCM stapling technology. The success and potential of this class of compounds has been already extensively demonstrated. Therefore, novel synthetic methodologies that expand the scope of the current approaches and grant easy access to stapled peptides continue to be of great interest in the field.. 11.

(30) 1.2.2 Cell Permeability of Stapled Peptides Over 95% of the peptides entering clinical trials in 2016 were directed at GPCRs and other extracellular targets, including receptor tyrosine kinases and ion channels, whereas only the remaining 5% were designed for intractable intracellular hosts62. A key factor for the low success in the latter group of peptide drugs is the overall poor cell permeability of these compounds. In this context, stapled -helical peptides have emerged as an effective solution to this problem, since when properly designed, they show significantly improved cell penetration capabilities 24-25, 30. . Several reports have investigated in depth the internalization of this class of compounds as well. as their mechanism of cellular uptake. Thus, Verdine’s laboratory developed an effective epifluorescence microscopy assay to evaluate the cell penetration of over 200 FITC-labeled peptides derived from several sequences and tethered with different staples that were placed at various positions in the parent sequence63. This quantitative study confirmed the substantial superior cell penetrating power of stapled peptides versus their linear counterparts, even when comparing them to natural unmodified cell penetrating sequences. This work also indicated that both the stapling type and the formal charge of the resulting macrocycle are key for peptide translocation, while other physical parameters didn’t have a significant effect. The former is especially favored by the incorporation of all-hydrocarbon staples and the latter by net positive charges below +7 at physiological pH. Interestingly, the correlation between cell penetration and net charge observed in this work is not in agreement with other studies carried out with linear peptides and mini-proteins, in which a higher net positive charge translates into better internalization likely due to higher interaction with the negatively charged phospholipid membrane. These researchers also provided mechanistic insights for the cellular uptake of such stapled. 12.

(31) peptides, which occurred by an endocytosis pathway and through the interaction with sulfated proteoglycans located in surface of the cell membrane. The Walensky laboratory has also investigated extensively both the biophysical parameters impacting the internalization of all-hydrocarbon stapled peptides and their import mechanism. Thus, in an early work carried out on leukemia cells, this group showed that BID BH3 stapled peptides were internalized through a micropinosomal mechanism followed by pinosomal release 19. This result was consistent with a later report that used electron microscopy assays to confirm vesicular cellular uptake, without plasma membrane disruption 64. The latter mechanism has also been demonstrated by high resolution fluorescence correlation microscopy studies, performed with stapled p53 peptides capable of restoring p53 function by dissociating both p53-HDM2 and p53HDMX interactions dose-dependently27. Likewise, an extensive study published by this laboratory in 2016 focused on determining the biophysical parameters favoring the cell penetration properties of these macrocycles65. This report studied the internalization of a staple-scanning library of FITClabeled stapled peptides designed to target BCL-X, an anti-apoptotic transmembrane protein that regulates mitochondrial biomolecules involved in programmed cell death mechanisms. The results identified the degree of hydrophobicity of the peptide as a major factor for enhanced cellular uptake, which is particularly favored when the hydrophobic all-hydrocarbon brace is placed at the amphipathic boundary of the peptide binding face. Notably, an excess of hydrophobicity and/or net positive charge are also undesired since cyclopeptides with such properties are more prone to cause membrane lysis at high concentrations. To date most of the comprehensive permeability studies have been carried out with allhydrocarbon stapled peptides. It is likely that the rules governing the cell penetrating power of such compounds also apply to the internalization of macrocycles bearing other types of staples.. 13.

(32) This hypothesis has been partially demonstrated by Zigang Li et al. who investigated the impact of different stapling architectures on the physicochemical and cell permeability properties of a model peptide, targeting the estrogen receptor coactivator 66. Notably, the authors observed that polar linkers such as lactam or triazole diminished substantially the cellular uptake, whereas hydrophobic braces improved significantly cell permeability. Overall, they concluded that hydrophobicity correlates well with peptide translocation, in contrast with helical character that was not a major factor. More comprehensive studies using other model peptides and staples with different chemistries will contribute to a better understanding of the biophysical determinants for the effective cellular uptake of stapled peptides. 1.2.3 Staple Peptide’s Stability to Proteolysis The ability of stapled peptides to evade protease degradation is one of the most beneficial properties validated for this class of peptidomimetics. Although stapling is typically applied to induce a desired bioactive -helical conformation67676767, such conformational restriction also limits the ability of enzymes to recognize their natural peptidyl substrate. By “blinding” proteases through macrocyclizations or with the incorporation of unnatural building blocks, in vitro proteolysis is decreased and serum stability is significantly improved 22, 68. This translates into enhanced pharmacokinetic properties and thus, into a more favorable drug profile. Indeed, the strong resistance of several families of stapled peptides to proteolytic degradation has been substantially validated in vitro, by subjecting them to enzymatic reactions using proteases with broad substrate specificity such as α-Chymotrypsin, Pepsin and Proteinase K60, 67. Likewise, their stability in plasma, liver microsomes and in vivo has also been well-demonstrated69. All these studies have shown that the lifetime of stapled peptides usually ranges from several hours to days, far exceeding that of their linear counterparts, which are mostly degraded within minutes. This. 14.

(33) remarkable resistance to proteolytic degradation has been observed in stapled peptides derived from large sequences and in macrocycles tethered by staples of various chemical nature. A representative example of the former was reported by Walensky and coworkers who introduced two all-hydrocarbon braces via the RCM reaction into a 36-residue long peptide to afford a doublestapled or “stitched peptide” (SAH-gp41), which effectively targets the HIC-1 fusion apparatus70. Notably, this compound showed enhanced stability both in vitro and in vivo and improved oral bioavailability, as compared to its linear parent sequence 22. As mentioned before, macrocycles stapled via methodologies other than RCM reaction also show strong protection to proteases, indicating that this feature is not highly dependent on the structure of the brace used for ligation. Thus, cyclopeptides containing bis-triazole, vinyl sulfide, bi-aryl or aryl-tryptophan linkers have all shown significantly improved stability to protease degradation. The ultimate validation of the stability achieved by means of properly introducing a stapling linker into a bioactive peptide sequence is given by the development of the first stapled peptide drugs entering clinical trials. Aileron Therapeutics, founded in 2005, announced its first all-hydrocarbon stapled peptide (ALRN-5281) entering human Phase I trials in 2013, as a potent agonist of the growth hormone releasing factor (GRF) currently tested for orphan endocrine disorders. The same company has recently developed a second stapled peptide drug, ATSP-7041, which targets an intracellular interaction in between MDM2 and MDMX and thus is capable of activating the function of the human tumor suppressor transcription factor p53 in vitro and in vivo29,71. 1.2.4 Stapled Peptides: Improved Target Binding Affinity and Diversity of Binding Strategies The potential of stapled peptides as effective chemical modulators of intracellular proteinprotein interactions was first demonstrated by Walensky et al. in 200419. In this breakthrough. 15.

(34) discovery, these researchers demonstrated that introducing all-hydrocarbon braces into unfolded BH3 peptides induced -helical conformations in such sequences, which showed helicities of up to 90% in solution. Moreover, the resulting all-hydrocarbon stapled peptides were remarkably resistant to proteolytic degradation in both in vitro and in vivo studies, and in addition, exhibited in vitro nanomolar binding affinity for their BCL-2 family targets. These cell penetrant peptidomimetics, referred to as “stabilized” alpha-helix of BCL-2 domains (or SAHBs), were also able to trigger cellular stress and apoptosis in vivo. After this original report, a large number of research groups have applied the RCM technology, and also most of the newly-developed stapling approaches, to generate a wide array of potent inhibitors of intracellular and extracellular PPIs. These modulators of protein function have shown potential applications in several diseases including cancer, metabolic and infectious diseases as well as neurological disorders (Table 2). Thus, for example, the Walensky laboratory has identified a series of BH3 mimetics that directly bind to MCL-1, and drive cancer cells to caspase-dependent apoptosis72. Other critical cancer-related mechanisms that have been successfully targeted by all-hydrocarbon stapled peptides include the Wnt/Beta-catenin and the NOTCH signaling pathways73-74. Likewise, Pellegrini et al. have used the two-component Azideyne stapling reaction to develop a potent inhibitor of the Ctf4-DNA Polymerase α interaction28. Ctf4/AND-1, a core component of the replisome progression complex, is an adaptor protein that bridges the helicase with Polymerase α complexes, and thus plays a key role in DNA replication, repairs and chromosome segregation. Despite its biological relevance, Ctf4 has been an elusive target possibly because it lacks a well-defined active site, being thus unfit for small molecule design. Notably, these investigators have successfully developed an (i, i+6) macrocycle (Sld5) that shows sub-micromolar binding affinity to Ctf4. Sld5 is capable of effectively displacing the Ctf4. 16.

(35) partner, DNA polymerase, and thus acts as a potent DNA-damaging cancer therapeutic agent. It is worth noting that although this cyclopeptide shows poor cellular uptake, it is expected that further modifications to the topology of the tethering linker will contribute to improve its overall cell permeability. The incorporation of a “staple” into a linear peptide sequence is now a proven and powerful tool for inducing bioactive α-helical conformations, and thus for the rational design of targeted peptide therapeutics. Interestingly, the spacer used for ligation can also provide additional binding benefits that extend beyond precise structural mimicry. This idea has been introduced by Popowicz and coworkers73, 75 who reported an X-ray structure of a stapled p53 peptide, SAH-p53-8, in complex with its protein target MDM273, 75. This structural analysis revealed that the hydrocarbon brace of SAH-p53-8 contributes to the binding energy by establishing hydrophobic interactions with some residues in the protein surface, thus expanding the role of the staple from a purely structural stabilizer or permeability enhancer, to an element that can be exploited for increased binding affinity. Overall, the progress achieved in the stapled peptide technology has allowed the identification of novel therapeutic agents targeting intractable protein surfaces, inadequate to be tackled by small molecule ligands. Such success is illustrated by the discovery of the first staple peptides entering human clinical trials. ALRN-6924, a second-generation macrocycle developed by Aileron Therapeutics, effectively disrupts the interaction between the p53 tumor suppression protein and both the murine double minute 2 (MDM2) and murine double minute X (MDMX) proteins, and is currently being tested in patients with peripheral T-cell lymphoma (PTCL), acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) 71. The potential success of this. 17.

(36) compound, and of others currently in development, could certainly transform the future of PPI’s inhibitors development. Table 1.2 Applications of the Stapled Peptide Technology in a Variety of Human Diseases Disease Cancer BCL-2 Proteins. Protein Target. Stapling Method. Reference. BAK/SAHB/BAX MCL-2/BH3/BAX BAK/BID-BH3. RCM RCM RCM. MDM2/MDMX. p53/(HDM2/MDM2) p53/(MDM2/MDMX) p53/MDM2 p53/MDM2 p53/MDM2 SAHM1/NOTCH BCL-9/βCATENIN IRS1/p110α EED/EZH2 RAB25/FIP XIAP & cIAPs PARP/Wnt TNKS/Wnt SLD5/CTF4 ER-coactivator. RCM RCM RCM Azide-yne Thiol-ene RCM RCM RCM RCM RCM Azide-yne Azide-yne Azide-yne Azide-yne Thiol-yne. Walensky et al.19, 70 Walensky et al.67 Moldoveneau et al.76 Bautista et al.77 Chang et al.26 Popowicz et al.75 Spring et al.78 Chou et al.45 Moellering et al.73 Ye Wu et al.74 Hao, Y. et al. 79 Kim W et al.80 Moellering et al.81 Terrett et al.68 Spring, et al.58 Spring, et al.58 Spring et al.28 Zigang et al.66. HIV-1 CA protein/ NYAD-1 GP41 six-helix bundle HCV-E2/ CD81. RCM RCM RCM. Long, Y. et al. 23 Walensky et al. 22 Cui et al. 82. GLP-1. Dithiol bisalkylation. Weijun Shen et al.83. ABCA1. RCM. Sviridov et al. 84. Estrogen receptor Estrogen receptor. RCM Thiol-yne. Irving et al. 85 Zigang et al. 66. Conantokins mimetic/NMDAR Neurotensin receptor. RCM. Gajewiak et al 86. C-C bond activation (Suzuki) C-H bond activation (Pd(II)). Jieping Zhu et al.. Dithiol alkylation Disulfide Linkage. Kritzer et al 36 King et al. 88. NOTCH BETA-CATENIN IRIS PRC2 RAB25 Programed cell death Wnt Signaling Wnt Signaling DNA Replication Receptor-coactivator Infectious Diseases HIV-1: Gag, integrase GP41 HCV-E2 Diabetes Insulin releasement Metabolism ABCA1 transporter Estrogen Receptors. Neurological Disorders NMDA receptor Neurotensin receptor Neurotransmitter at synapses Others Autophagy Neurotoxic. Dipeptidyl peptidase III ATP-activated human P2X3 receptor Human Beclin 1 rAps III/Na+ Channels. 18. 87. Lavilla et al. 60.

(37) 1.3. Long Range Macrocyclization Methodologies Long-range macrocyclization strategies are one of the earliest methods used to successfully. modify peptide’s secondary structures (Figure 1.3.1). Macrocyclization differs from cyclization based on the relative length of residues to be joined or linked, relative to the length of the peptide. Thus, in general terms 12 or more membered rings are considered “macrocycles”. Due to their large size, and thus the ability to interact with extended surfaces, macrocyclic peptides are also ideal candidates for disrupting protein-protein interactions. Indeed, over the last several decades, macrocyclizations have attracted a great deal of attention by medicinal chemists 76.. Figure 1.3.1 Structural representation of long range macrocyclization.. The strategies used for macrocyclizing peptides are diverse and escape any simple classification scheme77. Most broadly, methods are categorized based on what functional regions of the peptide are involved in the eventual linkage. The possibilities include head-to-tail, head/tailto-side chain, or side chain-to-side chain macrocyclic strategies. Within these broad descriptions of overall architecture, one can subcategorize based on the reaction pathway or the class of the resulting product. Several reactions have been applied to achieve macrocyclizations. The most popular ones include lactamization, lactonization, the RCM reaction, transition metal-catalyzed cross couplings and the one-pot azide-alkyne click chemistry cycloaddition76,78-80. Lactamization is likely the most frequently utilized, since it can exploit the reactivity between head and tail, basic side chains and tail, acidic side chains and head, or basic and acidic side chains. Evidently,. 19.

(38) lactamization can be applied to nearly every architectural class, largely due to the diversity of amino acid side chains. When combined with the use of non-natural amino acids, orthogonal protecting group strategies, and biosynthetic pathways, the methods and potential products of macrocyclization increase in diversity. Thus, in addition to standard reactions above-cited, other chemistries applied to macrocyclizing peptides include oxadiazole grafting81, sulfur-mediated cyclizations77,. 82. ,. isocyanides and other multicomponent reactions77, 82, and intein-catalyzed S/N acyl transfer83. All these approaches have been extensively reviewed and include both internal modifications to the linear peptide, and external template modifications 76,. 77, 82. . It is worth noting that for any. macrocyclization, it is crucial to control the factors favoring such transformation over other intermolecular reaction pathways. One highly effective strategy to achieve such a goal is by reducing the concentration of the reacting species, i.e, to perform reactions at high dilution and/or to control the rate of addition of either the substrate or reagents. Similarly, through conformational control of the linear precursor it is possible to lower the entropy of the substrate, and thus induce a “turned conformer” in which both reacting ends are brought in close spatial proximity. These strategies are also discussed in the previously mentioned reviews.. 1.3.1 Cell Permeability of Macrocyclic Peptides Cell permeability remains a major challenge for macrocyclic peptides. While they have been demonstrated as highly effective and specific modulators of many extracellular PPIs, few macrocyclic peptides demonstrate high cell permeability. A recent review by Walport and colleagues discusses potential paths forward for transforming macrocyclic peptides into cell permeable molecules84. Unlike -helices, where the characteristic secondary structure allows for. 20.

(39) the internalization of hydrogen bond-participating side chains, the secondary structures assumed by macrocyclic peptides do not necessarily possess this same helpful characteristic. Instead, the method of using N-methylated residues is frequently employed to reduce the number of hydrogen bond donors and increase overall hydrophobicity of macrocyclic peptides to increase cell permeability85 and bioavailability85-86. A novel alternative strategy for conferring cell permeability to macrocyclic peptides, involves coupling them with known cell permeable peptides (CPPs). Using this method, Trinh and co-workers tested the impact of incorporating cyclo(FΦRRRRQ) (cFΦR4, where Φ is L-2naphthylalanine), a CPP with known cell internalization properties, into macrocycles designed to target K-Ras, an oncogene that plays a key role in cell differentiation and apoptosis 87. Indeed, this strategy yielded cell-penetrating bicyclic peptides capable of inducing apoptosis in cancer cells, thus validating the applicability of this approach. The CPP used in this study is thought to gain intracellular access by binding directly to phospholipids of the cellular membrane and internalizing via endocytosis88.. 1.3.2 Macrocyclic Peptides as inhibitors of PPIs Macrocyclic peptides have been effective at targeting PPIs involved in a variety of biological pathways, including cancer, infectious and autoimmune diseases and regenerative medicine. Some representative applications of such inhibitors are presented in Table 1.3. An elegant and highly novel example showing the potential of this class of compounds have been recently published by Ito and co-workers89. Notably, the authors have developed dimeric macrocyclic peptides capable of inducing dimerization of the hepatocyte growth factor (HGF) receptor, also known as Met or c-Met, and thus activate downstream Met signaling cascades. These compounds underwent a first macrocyclization by a head-to tail thioether-linkage forming reaction, 21.

(40) between an N-terminal chloroacetyl group and thiol functionality. Dimerization of the monomers was next achieved by coupling C-terminal cysteine residues with bis-maleimide linkers of different lengths. The resulting dimeric macrocycles were capable of promoting cellular responses such as migration, proliferation and branching morphogenesis at low nanomolar concentration. This example demonstrates the unique cellular mechanisms accessible to macrocyclic peptide therapeutics. Table 1.3 Representative examples of macrocyclic peptides disrupting biologically relevant PPIs. Disease / Pathway. Protein Target. Macrocyclization Method. Reference. Leukemia. MM401-WDR5. RCM. Dou, Y. et al.92. Leukemia. MM589-WDR5. Lactamization (Internal). Wang, S. et al.. Ebola. VP24-KPNA5. Thioether. Suga, H. et al.102. Wound healing. Met-hHGF. Thioether. Suga, H. et al.. 103. PB1m6-Sema4D. Thioether. Suga, H. et al.. 104. Phosphorylation. AKT2-Pakti L1. Thioether. Suga, H. et al.105. Cancer. Ras-Cyclorasin 9A5. Lactamization (Internal). Pei, D. et al.. 106. Phosphorylation. PTP1B lactamization. Pei, D. et al.. 107. Immune response. 93. Cancer, Organogenesis. Cis−trans isomerization. Pin1. Identification and Intein-catalyzed S, N acyl isolation of potent. Fasan, R. et al.. Streptavidin. 96. transfer Streptavidin binder. 1.4. Peptidomimetic Molecules for Therapeutic Development: Significance Unquestionably, the methodologies applied to peptide stabilization have made a significant. contribution to therapeutic development. In particular, the discovery of the stapled peptide 22.

(41) technology by Verdine and colleagues in 2000 has triggered an exponential and growing number of relevant publications in the field. Considerable progress has been made in the synthetic approaches allowing access to these compounds. Likewise, the data generated so far have enhanced our overall understanding of basic rational design principles, mechanisms of cellular uptake and factors impacting the pharmacokinetic properties of these macrocycles. More importantly, this technology has allowed the identification of potent inhibitors of biologically relevant PPIs that were inaccessible using traditional medicinal chemistry approaches. Such remarkable advances, achieved in a relatively short period of time, have led to the development of the first stapled peptides entering human phase 1/2 clinical trials by Aileron Therapeutics, the leading company in the field, whereas several others compounds are currently in the pipeline. Altogether, these studies demonstrate the tremendous potential of peptidomimetic molecules for the generation of novel and effective therapies to treat incurable human diseases. In this regard, the work described in these doctoral thesis focuses, first, on developing a new and an efficient synthetic methodology for the chemoselective solid phase synthesis of singleand double-turn bis-thioether stapled peptides. The scope of this approach is demonstrated by synthesizing macrocycles derived from several sequences, validating its compatibility with different amino acids and SPPS. This method allows access to cyclopeptides tethered by allhydrocarbon linkers of different lengths, avoiding the use of unnatural amino acids and expensive catalysts. Moreover, it affords macrocycles with marked resistance to proteolytic degradation. We expect this feature to result in an improved pharmacokinetic profile that makes this family of compounds suitable for use in biological studies. Furthermore, we also present the application of our synthetic approach to generate stapled peptides that effectively disrupt biologically relevant intracellular PPIs. More specifically, we. 23.

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